PROJECT SUMMARY Most human pathogens enter via mucosal routes. Yet, there are few licensed mucosal vaccines. Thus, there is a need for new vaccine technologies and/or adjuvants that stimulate protective immunity at mucosal sites. We have developed a novel recombinant mucosal vaccine platform. The efficacy of this vaccine platform has been demonstrated using a human Fc?RI (hFc?RI)-specific fusion protein (FP) consisting of pneumococcal surface protein A (PspA) antigen (Ag) targeted to hFc?RI. This FP enhances immunogenicity and protection against mucosal S. pneumoniae (Sp) challenge when administered without adjuvant intranasally (i.n.) to hFc?RI transgenic mice. We hypothesize that a more potent adjuvant-free mucosal vaccine platform can be produced by generating a dual human FcRn (hFcRn)/hFc?RI-targeted-Ag FP, which enhances FcRn-mediated transepithelial Ag transport to the nasal-associated lymphoid tissue (NALT) and subsequent hFc?RI-mediated Ag presentation and T cell activation within the NALT. In this regard, such a dual-targeted FP has been generated by adding a hFcRn targeting sequence to the hFc?RI-specific PspA-containing FP. This novel FP will now be evaluated for its ability to enhance protective efficacy, transepithelial transport of Ag to the NALT, and Ag presentation/T cell activation within the NALT utilizing a hFcRn/hFc?RI-expressing mouse model. Specifically, in Aim 1, we will identify the optimal (most protective) FP configuration and immunization regimen. The hFc?RI-targeted PspA FP or anti-hFcRn/hFc?RI-PspA FP, as well as non-targeted PspA, will be administered to wildtype, hFc?RI, hFcRn, or hFcRn/hFc?RI-expressing mice at varying doses i.n. in the presence and absence of MPLA adjuvant. Protection against Sp challenge, as well as PspA-specific T and B cell responses, and bacterial burden, will be measured. The optimal immunization regimen will be identified based primarily on superior protection, but also optimal B and T cell responses. In Aim 2, we will identify the functional mechanisms involved in the FP-enhanced immunity/protection, as well as evaluate the safety of FP vaccination. We will examine FcRn binding and transepithelial transport of FPs in vitro and in vivo, FP binding to hFcR on APCs, FP internalization by APCs, and the ability of FPs to induce DC maturation and FP- enhanced Ag presentation/T cell activation. Safety parameters to be assessed will include health following vaccination, histological changes, and FP trafficking to the brain. Importantly, the above Aims will be carried out by a uniquely qualified investigative team with extensive expertise in FcRn and Fc?RI biology, as well as vaccine development and testing. Ultimately, these studies will be the first major step in establishing this novel/innovative vaccine platform as a viable adjuvant-free approach to mucosal vaccination. Furthermore, given the strong focus of vaccine research today on adjuvant discovery, maximizing the potency of this adjuvant-free vaccine platform will be crucial in changing perceptions regarding the requirement for adjuvant.